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Analysis of Defined Combinations of Monoclonal Antibodies in Anthrax Toxin 1 Neutralization Assays and Their Synergistic Action 2
3 Miriam M. Ngundi1, Bruce D. Meade2, Stephen F. Little3, Conrad P. Quinn4, Cindi R. 4
Corbett5, Rebecca A. Brady1, and Drusilla L. Burns1 5 6
Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, 7 MD 208921, Meade Biologics, Hillsborough, NC 272782, US Army Medical Research 8
Institute of Infectious Diseases, Fort Detrick, MD 217023, National Center for 9 Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 10
Atlanta, GA 303334. National Microbiology Laboratory, Public Health Agency of 11 Canada, Winnipeg, MB, Canada5. 12
13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 *Corresponding Author 28 29 30 Drusilla Burns 31 CBER, FDA HFM-434 32 Building 29, Room 130 33 8800 Rockville Pike 34 Bethesda, MD 20892 35 Tel: 301-402-3553 36 Fax: 301-402-2776 37 e-mail: [email protected] 38 39 40 Running Title: Interplay Between Toxin Neutralizing Antibodies 41 42
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.05714-11 CVI Accepts, published online ahead of print on 21 March 2012
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44 Abstract 45 46
Antibodies against the protective antigen (PA) component of anthrax toxin play an 47
important role in protection against disease caused by Bacillus anthracis. In this study, 48
we examined defined combinations of PA-specific monoclonal antibodies for their ability 49
to neutralize anthrax toxin in cell culture assays. We observed additive, synergistic and 50
antagonistic effects of the antibodies depending on the specific antibody combination 51
examined and the specific assay used. Synergistic toxin neutralizing antibody 52
interactions were examined in more detail. We found that one mechanism that can lead 53
to antibody synergy is the bridging of PA monomers by one antibody with resultant 54
bivalent binding of the second antibody. These results may aid in optimal design of new 55
vaccines and antibody therapies against anthrax. 56
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Inhalation anthrax, caused by the gram-positive bacterium Bacillus anthracis, is a 68
disease that is associated with high rates of morbidity and mortality if not treated early. 69
The potential use of B. anthracis spores as a biological warfare and bioterror agent has 70
spurred significant efforts towards the development of countermeasures for anthrax (16) 71
including new generation anthrax vaccines and therapeutics. Most anthrax vaccines and 72
therapeutic antibodies that are currently under development are designed to protect 73
against disease by targeting anthrax toxin, a major virulence factor of B. anthracis that is 74
believed to play a critical role in disease progression (27, 36). 75
Anthrax toxin is a tripartite toxin comprising protective antigen (PA), lethal factor 76
(LF) and edema factor (EF). PA combines with LF and EF to form lethal toxin (LT), 77
and edema toxin (ET), respectively (2, 8, 11, 17). Anthrax toxin is believed to be 78
important for outgrowth and trafficking of the bacteria during disease as well as the 79
progression and lethal nature of the disease (2, 10, 12, 19, 25, 27, 36). Because PA is a 80
common component of both ET and LT, most new anthrax vaccines and antibody 81
therapies target PA specifically (9, 14). Anti-PA antibodies have been shown to 82
neutralize anthrax toxin in vitro and confer protection in various animal models (13, 20, 83
21, 31, 41, 42) with levels of neutralizing antibodies correlating with protection (21, 35, 84
41). For this reason, assessment of toxin neutralization will likely play an important role 85
in the evaluation of new PA-based vaccines and therapeutic antibodies. 86
Evidence suggests that interplay can occur between antibodies against bacterial 87
toxins as they neutralize their target antigen. In a study of the neutralization of botulinum 88
toxin by monoclonal antibodies (mAbs), Nowakowski and colleagues demonstrated that a 89
combination of mAbs resulted in synergistic neutralization of that toxin. In that study, 90
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although no single mAb effectively neutralized the toxin, combinations of three mAbs 91
resulted in significant neutralization both in vivo and in vitro (30). Those results suggest 92
that a good understanding of the interplay that might occur between anti-PA antibodies as 93
they neutralize their target antigen could provide valuable information for optimal design 94
of antibody therapies and new vaccines against anthrax. 95
Toxin neutralization by a mixture of antibodies would be expected to be complex 96
in that neutralization depends, at least in part, on the array of epitopes recognized by the 97
antibodies, the binding affinities of the antibodies, the immunoglobulin classes present, 98
and any interactions that may occur between the antibodies and components of the 99
toxin’s target cell, e.g., Fcγ receptors (1, 7, 26, 34, 39, 40). While some anthrax toxin 100
neutralizing antibodies act exclusively by directly interfering with a critical aspect of 101
toxin action, other antibodies neutralize anthrax toxin by a mechanism that includes an 102
Fcγ receptor-mediated component (1, 28, 40). Another class of anti-PA antibody has 103
been described that enhances LT-mediated cytotoxicity through an Fcγ-receptor 104
dependent mechanism (24, 28). 105
Additive, synergistic, or even antagonist interactions might be expected to occur 106
between anti-PA antibodies present in a defined mixture of anti-PA monoclonal 107
antibodies or between antibodies induced by vaccination with PA-based vaccines. In 108
order to better understand the interplay that may occur between anti-PA antibodies, PA 109
and target cell components, we evaluated toxin neutralization using both individual anti-110
PA mAbs and combinations of those antibodies. In this study, we examined partially 111
neutralizing, fully neutralizing and toxicity-enhancing mAbs in cell culture assays, using 112
cell types that either do or do not express Fcγ receptors, to determine whether the 113
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interplay between the antibodies, PA and the target cell can result in additive, synergistic 114
and/or antagonistic effects. 115
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MATERIALS AND METHODS 117
Monoclonal Antibodies. AVR1046 was prepared in a similar manner to that previously 118
described by Boyer et al. (3). Briefly, 8-10 week old BALB/c mice were immunized 119
subcutaneously with 100 µg of anthrax recombinant PA adjuvanted with RIBI (Ribi 120
ImmunoChem Research, Inc., Hamilton, MT). Booster doses were given on days 21 and 121
35. On day 38, spleens were harvested and primary splenocytes isolated. Splenocytes 122
were fused with the mouse myeloma cell line SP 2/0 at a ratio of 1:5 123
(myeloma:splenocytes) in presence of PEG 4000 (Sigma, St. Louis, MO) and treated as 124
described previously (3). Cell culture supernatants were screened for anti-PA antibodies. 125
Anti-PA producing hybridomas were subcloned three times for isolation of antibody 126
producing cells. Generated mAbs were further screened for their ability to neutralize LT 127
activity in J774A.1 cell-based assay (18). F20G75 and 2F9 were prepared and 128
characterized as described by Gubbins et al. (15) and Little et al. (22), respectively. B. 129
anthracis Protective Antigen Antibody 18720 (C3), subsequently referred to in this report 130
as C3, was purchased from QED Bioscience, Inc. (San Diego, CA). 131
132
Reagents. Anthrax recombinant PA (NR-140 and NR-164), recombinant LF (NR-142), 133
recombinant EF (NR-2630) and murine macrophage-like J774A.1 cells (NR-28) were 134
from the NIH Biodefense and Emerging Infections Research Resources Repository, 135
NIAID, NIH (Bethesda, MD). The PA used in this study was verified by sodium 136
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dodecyl sulfate polyacrylamide gel electrophoresis to be > 95% full length. Epithelial-137
like CHO-K1 cells were purchased from American Type Culture Collection (ATCC, 138
Manassas, VA). Rat anti-mouse CD16/CD32 clone 2.4G2 was obtained from BD 139
Pharmingen (Franklin Lakes, NJ). 140
141
Toxin neutralizing antibody (TNA) assays. J774A.1 cells were cultured in Dulbecco’s 142
modified Eagle media (DMEM) containing 4.5 g/l D-glucose, 110 mg/l sodium pyruvate 143
and supplemented with 5% heat-inactivated bovine serum, 2mM L-glutamine, penicillin 144
(25 units/ml), streptomycin (25 µg/ml) and 10mM HEPES. The J774A.1 cell-based TNA 145
assay was performed as previously described (29). Briefly, cells were grown for 72 or 96 146
hours in culture flasks at 37oC, 5% CO2 and 95% relative humidity. The cells were 147
harvested, seeded in 96-well tissue culture plates (40,000 cells/well) and incubated for 148
17-19 hours. mAbs samples were prepared in a separate 96-well microtiter plate at two-149
fold dilutions and stored overnight at 4oC. For assays in which a combination of 150
antibodies was studied, one mAb was serially diluted starting with a concentration 151
approximately equal to its effective concentration at 50% inhibition (EC50), then the 152
second mAb, at a constant concentration, was spiked into each of the serial dilutions. 153
The spiking concentration was approximately equal to the EC50 of the second mAb. The 154
mAb samples were then incubated with a constant concentration of LT (50 ng/ml PA, 155
NR-140 and 40 ng/ml LF) for 30 minutes prior to adding to the cells. The cell-mAb-156
toxin mix was incubated for four hours after which 25 µl per well of 5 mg/ml tetrazolium 157
salt, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) were added. 158
After a two hour incubation, the cells were lysed using 100 µl per well of acidified 159
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isopropanol [90% isopropanol, 0.5% SDS (w/v) and 38 mM HCl] and the optical density 160
(OD) determined at 570 nm, with 690 nm as a reference filter. For assays performed with 161
the Fcγ-receptor blocking mAb 2.4G2, cells were pre-incubated with 100 µl of 10 µg/ml 162
mAb 2.4G2 for 15 min prior to addition of the mAb-toxin mix. mAb 2.4G2 remained on 163
the cells during the intoxication step. 164
CHO cells were grown in Kaighn’s modified F-12 nutrient mixture containing L-165
glutamine and supplemented with 10% heat-inactivated bovine serum, 2mM L-166
glutamine, penicillin (25 units/ml) and streptomycin (25 µg/ml). The CHO cell-based 167
TNA assay was performed as previously described (29). Briefly, cells and mAb samples 168
were treated as in the J774A.1 cell-based assay, except that the plated cells were 169
incubated for approximately 22 hours before addition of the mAb-toxin mix and that ET 170
(50 ng/ml, PA NR-140 and 160 ng/ml EF) was used instead of LT. To prevent cAMP 171
degradation, the mAb-toxin mix contained 750 µM of 3-isobutyl-1-methylxanthine. 172
After the four hour incubation, cells were washed three times with medium and cAMP 173
was estimated using the Tropix® chemiluminescent cAMP ELISA kit (Applied 174
Biosystems, Foster City, CA) as per the manufacturer’s instructions. The ELISA output 175
was measured as relative luminescence units (RLU) with one second integration. Since 176
this is a competitive assay, the measured RLU values are inversely proportional to the 177
amount of cAMP produced by the cells and therefore higher RLU values reflect greater 178
toxin neutralization. 179
180
Competitive Enzyme-Linked Immunoabsorbent Assay (ELISA). Ninety six-well 181
plates (Maxisorp, Nalge Nunc International, Rochester, NY) were coated with 1 µg/ml 182
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PA (NR-164) in PBS at 100 µl/well, overnight at 4oC. Meanwhile, serial dilutions of PA 183
(NR-164) containing biotinylated mAb AVR1046-IgG (0.13 pmol/ml) or biotinylated 184
AVR1046 Fab fragments (3.4 pmol/ml) in the presence and absence of mAb 2F9 (0.73 185
pmol/ml) in diluent buffer (1X PBS, pH 7.4 containing 0.5% Tween-20 and 5% non-fat 186
dry milk) were prepared in a separate plate and stored overnight at 4oC. PA-coated plates 187
were washed three times with wash buffer (1X PBS, pH 7.4 containing 0.1% Tween-20, 188
v/v), 100 µl/well of the PA-antibody samples were transferred to the coated plates and 189
incubated for one hour at 37oC. The plates were washed three times with wash buffer 190
and 100 µl per well goat anti-biotin IgG conjugated to horseradish peroxidase (Cell 191
Signaling Technology, Danvers, MA) were added. The plate was incubated for one hour 192
at 37oC, washed three times and 100 µl per well 2,2-azinobis(3-193
ethylbenzthiazolinesulfonic acid (ABTS) (KPL, Gaithersburg, MD) were added for color 194
development. After a 30 minute incubation at 37oC, 100 µl per well of ABTS® 195
Peroxidase Stop Solution (KPL, Gaithersburg, MD) were added and the optical density 196
(OD) was read at 405 nm with 490 nm as a reference filter. 197
198
Mapping of mAb binding to PA. The individual protein domains of PA, which are 199
composed of amino acids 1-258 (Domain 1), 259-487 (Domain 2), 488-595 (Domain 3), 200
and 596-735 (Domain 4) were cloned into E. coli strain BL21, expressed, and purified as 201
described previously (4). Each recombinant domain was resolved by SDS-PAGE and 202
transferred to nitrocellulose for immunoblotting. The blots were blocked with 5% non-203
fat dry milk in 10mM TBS, pH 7.3 containing 0.1% Tween-20, v/v, and then probed with 204
each monoclonal antibody at an appropriate dilution. Sheep anti-mouse IgG-HRP (GE 205
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Healthcare, Piscataway, NJ) was utilized as a secondary antibody, and reactivity was 206
visualized using a chemiluminescent substrate (Amersham ECL; GE Healthcare). 207
208
Fab fragment preparation and biotinylation of IgGs and Fab fragments. AVR1046-209
Fab fragments were prepared from AVR1046-IgG using Pierce Mouse IgG1 Fab and 210
F(ab′)2 preparation kit (Pierce, Rockford, IL) according to the manufacturer’s 211
instructions. Additional purification was performed using FPLC with a Superdex™ 200, 212
10/300 GL column (GE Healthcare Biosciences, Pittsburgh, PA). Purified Fab fragments 213
were concentrated and stored in 1X PBS at 4oC. 214
AVR1046-IgG and AVR1046-Fab fragments were biotinylated using EZ-Link® 215
NHS-LC-Biotin (Pierce, Rockford, IL). The IgG or Fab samples were incubated with 216
five-fold molar excess biotin in 50 mM bicarbonate buffer, pH 8.5 for 30 minutes. 217
Labeled IgG was separated from unincorporated biotin by size-exclusion chromatography 218
using a BioGel P10 column (BioRad, Hercules, CA) with UV-Vis detection at 280 nm. 219
Labeled Fab fragments were purified using a desalting column (Pierce, Rockford, IL). 220
Biotinylated IgG and Fab fragments were concentrated to approximately 1 mg/ml and 221
stored in 1X PBS at 4oC. The concentrations of the biotinylated IgG and Fab fragments 222
were determined using UV-Vis absorbance at 280 nm, using extinction coefficients of 223
1.43 and 1.53, respectively. 224
225
Data and statistical analyses. For TNA assays, OD570 and RLU values for the cell-only 226
control, run on the same plate as the sample, were set to 100%. Percent viability and 227
RLU for samples were then calculated relative to the OD570 and RLU, respectively, for 228
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the cell-only control. All data were plotted using GraphPad PRISM 5 software 229
(GraphPad Software Inc., La Jolla, CA). The neutralizing activities of mAbs were 230
calculated using curve fitting analyses performed in GraphPad PRISM 5 software. 231
Specifically, a four-parameter logistic (4-PL) regression model was used to fit the percent 232
viability or percent RLU versus the concentration of the antibody. Reported EC50 is the 233
inflection point for each curve from this model that represents 50% inhibition for the 234
corresponding antibody. For data that did not exhibit a symmetrical sigmoidal shape, 235
bell-shaped dose response curves were used to draw a smooth curve through the data. 236
For data interpretation and discussion, synergistic neutralization was reported when 237
neutralization activity of a combination of two mAbs was greater than the sum of 238
neutralization activities of the individual mAbs at any given concentration. Similarly 239
additive neutralization was reported as the neutralization activity yielded by a 240
combination of two mAbs approximating the sum of the mAbs’ individual neutralization 241
activities at a given concentration. For competitive ELISA, OD405 readings for each 242
curve were normalized to the OD405 of its upper asymptote set as 100% and the curves 243
were then fitted using a non-linear 4-PL curve fit model. 244
245
RESULTS 246
Analysis of selected individual mAbs in TNA assays. Two TNA assay formats have 247
been widely used in both research and clinical studies to assess the ability of anti-PA 248
antibodies to neutralize anthrax toxin (1, 6, 15, 22-24, 28, 37, 40). The two formats are 249
the J774A.1 cell-based TNA assay and the Chinese Hamster Ovary (CHO) cell-based 250
TNA assay. These assays differ both in the cell substrate and in the particular toxin—LT 251
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or ET—used to assess neutralization of PA action. J774A.1 cells are murine 252
macrophage-like cells that express Fcγ receptors (33, 39). The TNA assay based on these 253
cells measures neutralization of the cytocidal activity of LT. CHO cells are epithelial 254
cells that do not express Fcγ receptors (32). The TNA assay that utilizes CHO cells 255
measures the ability of antibodies to neutralize ET-induced increases in intracellular 256
cAMP levels. 257
For this study, we screened twenty five mAbs for toxin neutralization in both 258
assays. We identified three categories of mAbs based on their neutralization behaviors. 259
The first category was mAbs which were non-neutralizing in both assays, the second was 260
mAbs which neutralize toxin in both assays, and the third was mAbs which were non-261
neutralizing in the J774A.1 cell assay but neutralizing in the CHO cell assay. For further 262
studies, we chose two mAbs from each of the second and third categories. 263
Figure 1 shows the neutralization curves in the two assays for two neutralizing 264
mAbs, AVR1046 and F20G75. AVR1046 is a murine IgG1 (18). We mapped the 265
epitope for this mAb to domain 4 (amino acids 596-735) of PA, the receptor binding 266
domain, using purified PA domains and immunoblot analysis as described in the 267
Materials and Methods (data not shown). F20G75 is also a murine IgG1; this mAb binds 268
to a loop region extending from amino acids 304-319 found in domain 2 of PA that is 269
believed to be involved in pore formation (15). In the J774A.1 cell-based assay (Figure 270
1A), EC50 values for F20G75 and AVR1406 were 0.1 pmol/ml and 1.7 pmol/ml, 271
respectively (geometric mean of three independent assays), indicating that F20G75 was 272
significantly more neutralizing than AVR1046 on a molar basis (P = 0.0004; unpaired t-273
test) in that assay. In the CHO cell-based assay (Figure 1B), the EC50 values were 1.7 274
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pmol/ml and 2.7 pmol/ml for AVR1046 and F20G75, respectively (geometric mean of 275
three independent assays). If one compares neutralization of the two mAbs in the 276
J774A.1 cell-based assay, F20G75 was 17 times more effective than AVR1046 on a 277
molar basis, but in the CHO cell assay, no significant difference in neutralization was 278
observed (P = 0.15; unpaired t-test). In order to determine whether Fcγ receptors, which 279
are present on J774A.1 cells but absent on CHO cells, may have played a role in the 280
striking difference in relative neutralization observed between the two antibodies in the 281
two different assays, we blocked the major Fcγ receptors (IIB and III) expressed by 282
J774A.1 cells using the Fcγ receptor-blocking antibody mAb 2.4G2 (38). As shown in 283
Figure 1C, when these Fcγ receptors were blocked, the EC50 value for AVR1046 was 1.7 284
pmol/ml (geometric mean of three independent assays) which was identical to the value 285
observed without blocking the same receptors, indicating that AVR1046 neutralization 286
has no Fcγ receptor-mediated component. In contrast, the EC50 value for F20G75 was 287
3.1 pmol/ml (geometric mean of two independent assays) which was significantly 288
different from the value obtained for the same mAb when the Fcγ receptors were not 289
blocked (P < 0.0001; unpaired t-test). This result indicates that Fcγ receptors play a 290
major role in the neutralization of toxin by F20G75 in the J774A.1 cell-based assay. 291
Figure 2 shows the neutralization curve for two mAbs, 2F9 and C3, belonging to 292
the category of mAbs that are non-neutralizing in our J774A.1 cell assay but partially 293
neutralizing in CHO cell assay. 2F9 is a murine IgG1 antibody (22), as is C3 294
(manufacturer’s literature). We have mapped binding of 2F9 to domain 3 (amino acids 295
488 to 595) of PA, which is involved in heptamerization, and binding of C3 to domain 4 296
of PA (amino acids 596-735) (data not shown). These mAbs (2F9 and C3) exhibited 297
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non-neutralizing behavior in the J774A.1 cell-based assay using our routine assay 298
conditions that include fully cytotoxic concentrations of LT (Figure 2A). Of note, others 299
have previously shown that when 2F9 is used in a modified form of the J774 assay in 300
which sublethal concentrations of LT are used, 2F9 was observed to increase cytotoxicity 301
(24, 28). However, in our assay, since we are using fully lethal concentrations of LT, we 302
would not expect to be able to observe such an enhancement of cytoxicity. When we 303
examined 2F9 and C3 in the CHO-cell based assay, both mAbs exhibited some 304
neutralizing activity (Figure 2B) with EC50 values of 0.2 pmol/ml and 0.8 pmol/ml, 305
respectively (geometric mean of four independent assays). Of note, however, neither 306
mAb exhibited complete protection regardless of the amount of mAb used, as manifested 307
by an upper asymptote of the neutralization curve of less than 100% RLU. The 308
neutralizing capacity of C3 reached a plateau at a lower RLU value compared to that of 309
2F9. While 2F9 and C3 did not exhibit measurable neutralization in the J774A.1 based-310
assay, blocking Fcγ receptors of the cells renders the mAbs partially neutralizing (Figure 311
2C). The neutralization curves are not typical concentration-dependence curves, but 312
rather cell viability initially increased with increasing antibody concentration and then 313
decreased at higher antibody concentrations. While we do not know the reason for the 314
biphasic nature of the neutralization curves, one possibility is that Fcγ receptors I and IV 315
may play a role in the decrease in neutralization seen at the higher mAb levels, since 316
these Fcγ receptor types are not blocked by the Fcγ-receptor blocking antibody 317
mAb2.4G2 (38). Perhaps only at higher concentrations of 2F9 and C3 do sufficient 318
interactions between the mAbs and Fcγ receptors I and IV occur to lead to Fcγ receptor-319
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dependent enhanced toxicity involving these receptor types, resulting in the observed 320
drop in neutralization. 321
322
Analysis of combinations of mAbs in TNA assays. The production of different types of 323
mAbs (neutralizing, non-neutralizing and cytotoxicity-enhancing) against PA suggests 324
the likely presence of these diverse Abs in any given polyclonal antibody preparation. 325
Here we investigate the resultant neutralization exhibited by pairwise combinations of 326
mAbs. In order to not saturate neutralization, one mAb was serially diluted starting at a 327
concentration approximately equal to its EC50. The second mAb was then added, at a 328
constant concentration also approximately equal to its EC50, to the serial dilutions of the 329
first mAb. For comparison purposes, serial dilutions of the first mAb alone were also 330
assayed. 331
Figure 3 shows the neutralization of toxin by a combination of AVR1046 with 332
F20G75 (both neutralizing individually). When a mixture of F20G75 at its EC50 (0.13 333
pmol/ml) and increasing concentrations of AVR1046 was used to neutralize LT in the 334
J774A.1 cell-based assay, a decrease in cell viability was initially observed. As more 335
AVR1046 was added, the initial decrease was followed by an increase in cell viability 336
(Figure 3A). The minimum cell viability (dip) was observed at an AVR1046 337
concentration of 0.13 pmol/ml, a concentration equivalent to that of added F20G75. 338
Toxin neutralization by either AVR1046 or F20G75 alone did not exhibit such a decrease 339
in cell viability. A partially additive effect of the mAbs was observed at the higher 340
concentrations of AVR1046. When Fcγ receptors IIB and III were blocked, synergistic 341
neutralization was observed with the antibody combination when compared to the 342
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individual antibodies (Figure 3B), with no indication of the antagonistic effect that had 343
been observed when Fcγ receptors were not blocked. Please note, however, that because 344
the EC50 of F20G75 is greater when Fcγ receptors are blocked (3.1 pmol/ml; Figure 1), 345
F20G75 was used at a 10-fold higher concentration in this experiment than in the 346
experiment utilizing unblocked cells (0.1 pmol/ml; Figure 3A). 347
Combination studies of mAb (AVR1046) with mAb 2F9 were also conducted. 348
Synergistic toxin neutralization was observed in the J774A.1 cell assay for serial 349
dilutions of AVR1046 with a constant concentration of 2F9 (Figure 4A) or serial 350
dilutions of 2F9 with a constant concentration of AVR1046 (Figure 4B). Finally, we 351
examined the combination of mAbs 2F9 and C3. As described above, when assayed 352
individually in our J774A.1 cell-based assay, neither mAb exhibited neutralizing activity 353
(Figure 2A); however, in the CHO-cell based assay, they were both partially neutralizing 354
(Figure 2B). Surprisingly, the mAb combination showed a robust synergistic 355
neutralization of LT in the J774A.1 cell-based assay (Figures 5A and B). Synergy was 356
observed regardless of which mAb was serially diluted. In the CHO cell-based assay, 357
when the two mAbs were combined, neutralization appeared to be additive (Figures 5C 358
and D) regardless of which mAb was serially diluted. 359
360
Investigation of the mechanism underlying synergistic neutralization. Nowakowski 361
et al. (30) demonstrated that synergistic neutralization of botulinum toxin by multiple 362
antibodies was a result of an increase in functional binding affinity. Those investigators 363
suggested that an increase in functional binding affinity could be due to binding of one 364
IgG antibody to two toxin molecules, which could then favor bivalent binding of the 365
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second antibody with a resultant increase in antibody avidity, with avidity being the 366
combined strength of multiple bond interactions. Alternatively, binding of the first 367
antibody might induce or stabilize a conformation of the toxin that favors the binding of 368
the second antibody. Those investigators did not further investigate which of these 369
mechanisms might underlie the increase in functional binding that they observed. 370
We reasoned that similar mechanisms might be the basis for the synergy that we 371
observed between the PA mAbs. To investigate whether binding of 2F9 to PA could 372
“convert” PA to a multivalent antigen by bridging PA monomers—thereby facilitating 373
bivalent binding of AVR1046—we utilized AVR1046 Fab fragments in a competitive 374
ELISA and compared the results to those obtained using AVR1046 IgG. While IgG can 375
bind bivalently, Fab fragments are limited to monovalent binding. By comparing the 376
binding properties of the AVR1046 IgG and its Fab fragments, we would be able to 377
evaluate whether 2F9 induces bivalent binding of AVR1046 to PA molecules. 378
Because we believe that soluble PA better represents the biologically relevant 379
form of PA, for these experiments we used a competitive ELISA format, instead of the 380
normal indirect ELISA, in order to measure binding to PA in solution rather than to PA 381
bound to the plastic plate. In this competitive ELISA, serial dilutions of PA were 382
incubated with constant amounts of antibodies or Fab fragments overnight at 4˚C to allow 383
binding to reach equilibrium. The PA-antibody mixtures were then added to 96-well 384
plates that had been coated with PA. In order to assess AVR1046 binding in a manner 385
that distinguishes it from that of 2F9, the AVR1046 IgG and Fab fragments used in the 386
assay were biotinylated and detected in the assay using an anti-biotin HRP conjugate. In 387
this competition assay, one would expect that, as the concentration of soluble PA is 388
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increased, more of the AVR1046 would become bound to this species and therefore less 389
would be available for binding to the PA-coated plate. The IC50 (the concentration of 390
soluble PA that is required for 50% inhibition of AVR1046 binding to the PA coating the 391
plate) can be measured. If 2F9 increases the avidity of AVR1046 or AVR1046 Fab 392
fragments for the soluble PA, then the amount of soluble PA needed to prevent binding of 393
AVR1046 or AVR1046 Fab fragments to the PA-coated plate should decrease (i.e., the 394
IC50 for soluble PA would decrease). 395
Figure 6A shows that the concentration of soluble PA needed to prevent binding 396
of biotinylated AVR1046 IgG to the PA-coated plate was significantly less in the 397
presence of 2F9 than in its absence (i.e., the observed IC50 decreases in the presence of 398
2F9, reflective of an increase in the avidity of AVR1046 for soluble PA). In contrast 399
(Figure 6B), the amount of soluble PA needed to prevent binding of biotinylated 400
AVR1046 Fab fragments to the PA-coated plate was similar in the presence or absence of 401
2F9 (i.e., no change in IC50 for soluble PA, reflective of no change in the avidity of 402
AVR1046 Fab fragments for PA). Thus 2F9 increased the avidity of a form of AVR1046 403
that has two binding sites for PA but not a form that has only a single PA binding site. 404
This observation suggests that 2F9 can promote bivalent binding of AVR1046 IgG to PA, 405
presumably by bridging two PA monomers. 406
407
DISCUSSION 408
In the course of this study, two major findings emerged. First, assessment of the 409
neutralizing capacity of any particular antibody can be highly dependent on the TNA 410
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assay used. Second, the interplay between antibodies, PA, and any Fcγ receptors that 411
may be present can result in additive, synergistic, or antagonistic interactions. 412
The first important aspect of this work is our finding that different TNA assays 413
can give strikingly different impressions of antibody neutralization. As seen in Figure 1, 414
mAb F20G75 was significantly more neutralizing than AVR1046 in the J774A.1 cell-415
based assay, but the two antibodies exhibited approximately the same neutralizing 416
capacity in the CHO cell-based assay. We found that this difference was likely due, at 417
least in part, to fact that neutralization by F20G75 is highly dependent on Fcγ receptors 418
(Figure 1C). Because of this Fcγ-receptor dependence, very different impressions of the 419
neutralizing capacity of this mAb are given by the two different assays. These results 420
raise the question of which assay more accurately reflects antibody neutralization of 421
anthrax toxin in vivo. Pertinent to this question are the recent findings of Abboud et al. 422
who reported that passive immunization with an anti-PA mAb protected wild-type mice, 423
but not FcγR-deficient mice, against B. anthracis infection (1), suggesting that Fcγ 424
receptors do play a role in toxin neutralization or toxin clearance in vivo. While more 425
work is needed to make definitive conclusions concerning which assay is more relevant 426
to antibody neutralization in vivo, our work suggests that careful thought should be given 427
the choice of the assay when assigning and/or comparing the neutralization activities of 428
mAbs. 429
A second aspect of our study demonstrates that the interplay between antibodies, 430
PA and any Fcγ receptors that may be present on target cells can result in several 431
different types of interactions. Additive interactions between antibodies, which have 432
been reported previously for antibody binding to PA (5), were found and would be 433
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expected since PA is sufficiently large to bind to more than one antibody at a time. 434
While others have previously reported that one antibody directed to PA combined with 435
another directed to LF provided a synergistic protection in vivo (6), to our knowledge, 436
synergism between two PA antibodies has not been demonstrated previously. In our 437
study, we found several instances of synergy. 438
The combination of AVR1046 and 2F9 exhibited synergistic neutralization in the 439
J774A.1 cell assay (Figure 4). When we examined the molecular basis for the synergy 440
between these antibodies, we found that the binding of 2F9 promoted bivalent binding of 441
AVR1046 (Figure 6). Because full-length PA is normally found in the monomeric form 442
in solution, these results would suggest that each of these mAbs is capable of bridging PA 443
monomers. Bridging by one of the antibodies would promote bridging by the other. Any 444
transient dissociation of one antibody arm from the antigen would result in rapid 445
rebinding since the other antibody bridge would prevent the antigen from diffusing away. 446
This phenomenon would substantially increase antibody avidity resulting in synergistic 447
neutralization. 448
We also observed synergistic neutralization with the combination of AVR1046 449
and F20G75 in the J774A.1 cell assay (Figure 3), but only when the majority of Fcγ 450
receptors were blocked (i.e., in the presence of the Fcγ receptor-blocking antibody mAb 451
2.4G2). The neutralization pattern for this combination of antibodies in the presence of 452
Fcγ receptors was complex and will be discussed below. Because AVR1046 can bridge 453
two PA monomers, the mechanism underlying the synergy observed may be the same as 454
that discussed above for AVR1046 and 2F9, i.e., induction of bivalent binding. 455
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The combination of mAbs 2F9 and C3 yielded what was perhaps the most 456
surprising result. While neither antibody exhibited any neutralization in our J774A.1 457
cell-based assay, when mixed together, significant neutralization was observed (Figures 458
5A and 5B). In contrast, this synergy was not observed in the CHO cell-based assay 459
(Figure 5C and 5D). The interactions between the two antibodies, PA, and possibly Fcγ 460
receptors that result in synergistic neutralization on J774A.1 cells remain to be 461
elucidated. 462
In our studies, we noted one instance of antagonistic interactions between 463
antibodies. When the combination of AVR1046 and F20G75 was examined in the 464
J774A.1 cell-based assay (Figure 3A), a complex pattern was noted, which was highly 465
dependent on Fcγ receptors. We observed that as the concentration of AVR1046 was 466
increased, an initial antagonism between AVR1046 and F20G75 was observed, as 467
manifested by a decrease in neutralization. As AVR1046 concentration was further 468
increased, neutralization gradually increased. Because neutralization by F20G75 is 469
highly dependent on Fcγ receptors, the initial dip in neutralization that was observed 470
could be explained if AVR1046 prevents the PA-F20G75 complex from binding to Fcγ 471
receptors. Such inhibition might be due to direct steric inhibition of the formation of a 472
F20G75-PA-Fcγ receptor complex by AVR1046. Alternatively, since AVR1046 binds to 473
the receptor binding domain of PA, this antibody may inhibit PA binding to its cell 474
surface receptor, thereby decreasing the effective concentration of PA at the cell surface. 475
This effective decrease in concentration would result in fewer opportunities for a PA-476
F20G75-Fcγ receptor complex to form. As the concentration of AVR1046 is further 477
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increased, neutralization by AVR1046 would be expected to become dominant, 478
consistent with the recovery in neutralization that was observed. 479
From the results of our study, we can conclude that additive, synergistic, or 480
antagonistic interactions can occur among anti-PA antibodies, PA, and Fcγ receptors that 481
may be present on the cell surface. We have demonstrated that one mechanism that can 482
lead to antibody synergy is the bridging of PA monomers in solution by one antibody, 483
with resultant bivalent binding of the second antibody. Our demonstration of anti-PA 484
antibody synergy suggests that the design of new anthrax antibody therapies and vaccines 485
might be better optimized if these findings are taken into account. For example, 486
appropriate combinations of mAbs, rather than individual antibodies alone, might result 487
in more favorable therapeutic outcomes. Specifically tailoring new vaccines to modulate 488
the polyclonal response in such as way as to promote synergistic neutralization, while 489
admittedly challenging, might be set as a future goal. In this study, we examined the 490
interplay between anti-PA antibodies exclusively; however, we believe that our findings 491
may apply broadly to neutralizing antibodies against many bacterial toxins. 492
493
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ACKNOWLEDGEMENTS 494
This work was supported in part by an interagency agreement between the 495
National Institute of Allergy and Infectious Diseases, NIH, and the Food and Drug 496
Administration. 497
The following reagents were obtained from the NIH Biodefense and Emerging 498
Infections Research Resources Repository, NIAID, NIH: anthrax LF, recombinant from 499
Bacillus anthracis, NR-142; anthrax PA, recombinant from Bacillus anthracis, NR-140; 500
anthrax PA, recombinant from Bacillus anthracis, NR-164; anthrax EF, recombinant 501
from Bacillus anthracis, NR-2630; and J774A.1 monocyte/macrophage (mouse) Working 502
Cell Bank, NR-28.503
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FIGURE LEGENDS 653
Figure 1. Concentration-dependence curves for the neutralization of protective antigen 654
by mAbs AVR1046 and F20G75 in the J774A.1 and CHO cell-based assays. The 655
indicated concentrations of mAb AVR1046 (●) and mAb F20G75 (■) were used to 656
neutralize a constant concentration of either LT in the J774A.1 cell-based assay (Panels A 657
and C) or ET in the CHO cell-based assay (Panel B). In Panel C, FcγRIIB/III receptors 658
were blocked by the addition of mAb 2.4G2 as described in Materials and Methods. 659
Each point corresponds to the mean of the values obtained for three independent sample 660
preparations, with the standard deviation (SD) indicated by the error bar. The samples 661
were run on duplicate plates for the J774.1A cell based assay and on a single plate for the 662
CHO cell-based assay. Each figure is representative of three independent assays run on 663
different days. 664
665
Figure 2. Concentration-dependence curves for the neutralization of protective antigen 666
by mAbs 2F9 and C3 in the J774A.1 and CHO cell-based assays. The indicated 667
concentrations of mAb 2F9 (●) and mAb C3 (■) were used to neutralize a constant 668
concentration of either LT in the J774A.1 cell-based assay (Panels A and C) or ET in the 669
CHO cell-based assay (Panel B). In Panel C, FcγRIIB/III receptors were blocked by the 670
addition of mAb 2.4G2 as described in Materials and Methods. Each point corresponds 671
to the mean of the values obtained for three independent sample preparations, with the 672
standard deviation (SD) indicated by the error bar. The samples were run on duplicate 673
plates for the J774.1A cell based assay and on a single plate for the CHO cell-based 674
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assay. Each figure is representative of three independent assays each run on different 675
days. 676
677
Figure 3. Concentration-dependence curves for toxin neutralization by the combination 678
of mAbs AVR1046 and F20G75. The concentration of one antibody was varied in the 679
manner indicated on the x-axis while that of the other was held constant. Cell viability is 680
indicated for concentrations of the serially-diluted antibody assayed either individually 681
(●) or combined (■) with the other mAb held constant at the concentration indicated in 682
the panel. Neutralization obtained with the antibody that was held constant, in the 683
absence of the serially diluted antibody, is indicated on the y-axis (▲). In Panel B, 684
assays were conducted in the presence of the Fcγ receptor-blocking antibody mAb 2.4G2 685
as described in Materials and Methods. Each point corresponds to the mean of the values 686
obtained for three independent sample preparations run on the same plate, with the 687
standard deviation (SD) indicated by the error bar. For each independent assay, the 688
samples were run on duplicate plates. Each figure is representative of the independent 689
assays each run on at least three different days. 690
691
Figure 4. Concentration-dependence curves for toxin neutralization of protective antigen 692
by the combination of mAbs AVR1046 and 2F9. The concentration of one antibody was 693
varied in the manner indicated on the x-axis while that of the other was held constant. 694
Cell viability is indicated for concentrations of the serially-diluted antibody assayed 695
either individually (●) or combined (■) with the other mAb held constant at the 696
concentration indicated in the panel. Neutralization obtained with the antibody that was 697
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held constant, in the absence of the serially diluted antibody, is indicated on the y-axis 698
(▲). Each point corresponds to the mean of the values obtained for three independent 699
sample preparations, with the standard deviation (SD) indicated by the error bar. For 700
each independent assay, samples were run on duplicate plates. Each figure is 701
representative of three independent assays each run on different days. 702
703
Figure 5. Concentration-dependence curves for toxin neutralization by the combination 704
of mAbs C3 and 2F9. The concentration of one antibody was varied in the manner 705
indicated on the x-axis while that of the other was held constant. Cell viability in the 706
J774A.1 cell-based assay (panels A and B) or % RLU in the CHO cell-based assay 707
(Panels C and D) is indicated for concentrations of the serially-diluted antibody assayed 708
either individually (●) or combined (■) with the other mAb held constant at the 709
concentration indicated in the panel. Neutralization obtained with the antibody that was 710
held constant, in the absence of the serially diluted antibody, is indicated on the y-axis 711
(▲). Each point corresponds to the mean of the values obtained for three independent 712
sample preparations, with the standard deviation (SD) indicated by the error bar. For 713
each independent assay, samples were run on duplicate plates. Each figure is 714
representative of three independent assays each run on different days. 715
716
Figure 6. Analysis of mAb AVR1046 binding in the presence or absence of mAb 2F9 717
using a competitive PA ELISA with soluble PA as the competitor. The indicated 718
concentrations of soluble PA mixed with either biotinylated AVR1046 IgG (B-719
AVR1046) in the absence (●) and presence (■) of 2F9 (Panel A) or B-AVR1046 Fab 720
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fragments in the absence (●) and in the presence (■) of 2F9 (Panel B) were assayed for 721
binding using the competitive ELISA format described in Materials and Methods. The 722
ability of soluble PA to inhibit binding of B-AVR1046 or B-AVR1046 Fab fragments to 723
the PA coating the plate was determined. The OD405 readings for each competition curve 724
were normalized to the OD405 of its upper asymptote set as 100% and the curves were 725
then fitted to non-linear 4PL curve fit. Each point corresponds to the mean of the values 726
obtained for three independent sample preparations run on the same plate, with the 727
standard deviation (SD) indicated by the error bar. Each figure is representative of the 728
three independent assays each run on different days. 729
730
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A J774 cells
Figure 1
J774 cells + 2 4G2C
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100
120A J774 cells
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120J774 cells + 2.4G2C
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ell v
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20F20G75AVR1046
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Figure 2
80
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100
2F9C3
J774 cellsA
80
100
2F9C3
J774 cells + 2.4G2C
y
40
60
% R
LU
20
40
60
% C
ell v
iabi
lity
40
60
% C
ell v
iabi
lity
0.01 0.1 1 10 1000
20
[mAb] pmol/ml0.01 0.1 1 10 100 10000
20
[mAb] pmol/ml0.01 0.1 1 10 100 10000
20
[mAb] pmol/ml on March 26, 2021 by guest
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Figure 3
120J774A.1 cells + 2.4G2B
120
J774 cellsA
80
100
iabi
lity
80
100
120
abili
ty
20
40
60
AVR1046 + F20G75 (1.3 pmol/ml)AVR1046
% C
ell v
i
20
40
60
AVR1046 + F20G75 (0.13pmol/ml)AVR1046
% C
ell v
ia
0.01 0.1 10
F20G75 (1.3 pmol/ml)
0//
[AVR1046] pmol/ml0.001 0.01 0.1 1
0
F20G75 (0.13pmol/ml)
0 //[AVR1046] pmol/ml
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100
J774 cellsB
Figure 4
100
J774 cellsA
60
80
100
l via
bilit
y60
80
100
via
bilit
y
0
20
40
2F9 + AVR1046 (2 pmol/ml)AVR1046 (2 pmol/ml)
//
2F9% C
ell
0
20
40
AVR1046 + 2F9 (0.4 pmol/ml)2F9 (0.4 pmol/ml)
//
AVR1046% C
ell
0.01 0.1 10 //0
[2F9] pmol/ml0.01 0.1 1 10
0 //0
[AVR1046] pmol/ml
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80
100J774 cellsB
Figure 5
80
100J774 cellsA
20
40
60
80
2F9 + C3 (0.67 pmol/ml)C3 (0 67 l/ l)
2F9
% C
ell v
iabi
lity
20
40
60
80
C3 + 2F9 (0.4 pmol/ml)2F9 (0 4 pmol/ml)
C3
% C
ell v
iabi
lity
0.01 0.1 10
C3 (0.67 pmol/ml)
//0[2F9] pmol/ml
0.01 0.1 1 100
2F9 (0.4 pmol/ml)
//0
[C3] pmol/ml
100CHO cellsD
100CHO cellsC
40
60
80
% R
LU
40
60
80
% R
LU
0.01 0.1 10
20 2F9 + C3 (0.67 pmol/ml)C3 (0.67 pmol/ml)
2F9
//0
[2F9] pmol/ml0.01 0.1 1 10
0
20C3 + 2F9 (0.4 pmol/ml)2F9 (0.4 pmol/ml)
C3
//0[C3] pmol/ml
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AVR1046 F b / 2F9B
Figure 6
AVR1046 I G / 2F9A
100
120
B AVR1046 Fab
AVR1046 Fab +/- 2F9B
Fab100
120
B-AVR1046 IgG
AVR1046 IgG +/- 2F9A
IgG
60
80
B-AVR1046 FabB-AVR1046 Fab + 2F9
of B
-AVR
1046
F
60
80
B-AVR1046 IgGB-AVR1046 IgG + 2F9
of B
-AVR
1046
I
20
40
% B
indi
ng o
20
40
% B
indi
ng o
0.0001 0.01 1 100 100000
[PA83] pmol/ml0.0001 0.01 1 100 100000
[PA 83] pmol/ml
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